Apoptag peroxidase kit

Assessing of apoptotic germ cell was performed in testicular sections by the terminal deoxynucleotidyl transferase (TdT)-mediated deoxy-UTP nick end labeling (TUNEL) technique [29 (link)] using an ApopTag-peroxidase kit (Chemicon International, Inc., Temecula, CA). Enumeration of the apoptotic germ cell population and Sertoli cell nuclei with distinct nucleoli was quantified at stages I-IV (early stages), stages VII-VIII (middle stages), and stages XI-XII (late stages) of the seminiferous epithelial cycle by using an Olympus BH-2 microscope (New Hyde Park, NY). Stages were identified according to the criteria proposed by Russell et al for paraffin sections [30 (link)]. The rate of germ cell apoptosis was expressed as the number of apoptotic germ cells per Sertoli cell (apoptotic index, AI) [29 (link)].

All animal experiments were approved by the Institutional Animal Care and Use Committee of the University of Pittsburgh. HCT116 cells (<70% confluent) were harvested, and 5×10⁶ cells in 0.1 mL of medium were implanted subcutaneously on the back of athymic nude female mice. When the tumor size reached approximately 100 mm³, mice were randomized into the following groups (5 mice per group): (A) control (vehicle; 5% dextrose); (B) 40 mg/kg, (C) 80 mg/kg and (D) 120 mg/kg CRT0066101 (dissolved in 5% dextrose) administered orally once daily. Therapy was administered for 3 weeks, and animals were sacrificed on day 21 after treatment with CRT0066101. Tumor volume was measured as V = 1/2ab², in which “a” and “b” represents length and width of tumor (22 (link)). Tumor volumes were monitored 3 times per week. At the time the animals were euthanized, half of the tumor tissue was fixed with formalin and paraffin-embedded for immunohistochemistry. The other half was snap-frozen in liquid nitrogen and stored at −80°C. Tissue slides were processed by the Department of Pathology Development Laboratory and the Tissue and Research Pathology Services at the University of Pittsburgh for Ki-67 (#9027; Cell Signaling), in situ TUNEL staining (APOPTAG Peroxidase kit; Chemicon), p-ERK (#4370; Cell Signaling), and M30 (#12140322001; Roche).

In situ detection of cells with DNA strand breaks was performed in formalin-fixed, paraffin-embedded liver sections by the terminal deoxynucleotidyl transferase (TdT)-mediated deoxy-UTP nick end labeling (TUNEL) technique [17 (link), 25 (link)] using an ApopTag-peroxidase kit (Chemicon International, Inc., San Francisco, CA, USA). The rate of hepatocellular apoptosis was expressed as the percentage of the TUNEL-positive apoptotic nuclei per total (apoptotic plus non-apoptotic) nuclei present within a reference area of 62,500 μm² [17 (link), 25 (link)].